Macrocyclic beta-secretase inhibitors

ABSTRACT

Disclosed are novel compounds of the formula 
                         
or a pharmaceutically acceptable salt or solvate thereof, wherein R 1 , R 2 , R 3 , n and X are as defined in the specification. Also disclosed are pharmaceutical compositions comprising the compounds of formula I. Also disclosed are methods of treating cognitive or neurodegenerative diseases such as Alzheimer&#39;s disease. Also disclosed are methods of treating a cognitive or neurodegenerative disease comprising administering to a patient I need of such treatment a combination of at least one compound of formula I and at least one compound selected from the group consisting of β-secretase inhibitors other than those of formula I, HMG-CoA reductase inhibitors, gamma-secretase inhibitors, non-steroidal anti-inflammatory agents, N-methyl-D-aspartate receptor antagonists, cholinesterase inhibitors and anti-amyloid antibodies.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of application U.S. Ser. No.11/189,346, filed Jul. 26, 2005, issued as U.S. Pat. No. 7,652,003 onJan. 26, 2010, which claims the benefit of priority to U.S. ProvisionalApplication No. 60/591,899, filed Jul. 28, 2004, each of which isincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to substituted macrocyclic BACE-1 inhibitors,pharmaceutical compositions comprising said compounds, and their use inthe treatment of Alzheimer's disease.

BACKGROUND

Alzheimer's disease (AD) is a progressive neurodegenerative disease thatis ultimately fatal. Disease progression is associated with gradual lossof cognitive function related to memory, reasoning, orientation andjudgment. Behavioral changes including confusion, depression andaggression also manifest as the disease progresses. The cognitive andbehavioral dysfunction is believed to result from altered neuronalfunction and neuronal loss in the hippocampus and cerebral cortex. Thecurrently available AD treatments are palliative, and while theyameliorate the cognitive and behavioral disorders, they do not preventdisease progression. Therefore there is an unmet medical need for ADtreatments that halt disease progression.

Pathological hallmarks of AD are the deposition of extracellularβ-amyloid (Aβ) plaques and intracellular neurofibrillary tanglescomprised of abnormally phosphorylated protein tau. Individuals with ADexhibit characteristic Aβ deposits, in brain regions known to beimportant for memory and cognition. It is believed that Aβ is thefundamental causative agent of neuronal cell loss and dysfunction, whichis associated with cognitive and behavioral decline. Amyloid plaquesconsist predominantly of Aβ peptides comprised of 40-42 amino acidresidues, which are derived from processing of amyloid precursor protein(APP). APP is processed by multiple distinct protease activities. Aβpeptides result from the cleavage of APP by β-secretase at the positioncorresponding to the N-terminus of Aβ, and at the C-terminus byγ-secretase activity. APP is also cleaved by α-secretase activityresulting in the secreted, non-amyloidogenic fragment known as solubleAPP.

An aspartyl protease known as BACE-1 has been identified as theβ-secretase activity responsible for cleavage of APP at the positioncorresponding to the N-terminus of Aβ peptides.

Accumulated biochemical and genetic evidence supports a central role ofAβ in the etiology of AD. For example, Aβ has been shown to be toxic toneuronal cells in vitro and when injected into rodent brains.Furthermore inherited forms of early-onset AD are known in whichwell-defined mutations of APP or the presenilins are present. Thesemutations enhance the production of Aβ and are considered causative ofAD.

Since Aβpeptides are formed as a result of β-secretase activity,inhibition of BACE-1 should inhibit formation of Aβ peptides. Thusinhibition of BACE-1 is a therapeutic approach to the treatment of ADand other cognitive and neurodegenerative diseases caused by Aβ plaquedeposition.

Substituted amine BACE-1 inhibitors are disclosed in, WO 04/04396, WO02/02505, WO 02/02506, WO 02/02512, WO 02/02518 and WO 02/02520. Renininhibitors comprising a (1-amino-2 hydroxy-2-heterocyclic)ethyl moietyare disclosed in WO 89/03842. WO 02/088101 discloses BACE inhibitorsfunctionally described as being comprised of four hydrophobic moieties,as well as series of compounds preferably comprising a heterocyclic orheteroaryl moiety.

WO 02/100856 and WO 02/100399 disclose macrocycles and methods forpreparing macrocycles useful in the treatment of Alzheimer's disease.

SUMMARY OF THE INVENTION

The present invention relates to compounds having the structural formulaI

or a pharmaceutically acceptable salt or solvate thereof, wherein

R¹ is

R² is —N(R⁵)C(O)R⁴— or heterocyclylene ring;

R³ is arylene, heteroarylene, heterocyclylene or cycloalkylene;

R⁴ is arylene, heteroarylene, heterocyclylene or cycloalkylene;

R⁵ is hydrogen, alkyl, aryl, heteroaryl or cycloalkyl;

R⁶ and R⁷ are independently selected from hydrogen, —OH, alkyl,cycloalkyl, cycloalkylalkyl, heterocyclyl, aryl, heteroaryl, aralkyl,heteroaralkyl, aralkoxy, heteroaralkoxy and alkoxy, with the provisothat when R⁶ and R⁷ are —OH, aralkoxy, heteroaralkoxy and alkoxy, R⁶ andR⁷ are not attached to a ring carbon adjacent to a ring nitrogen;

R⁸ is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl,aralkyl, heteroaralkyl, heterocyclyl, heterocyclylalkyl, —C(O)R⁹,—C(O)OR¹², —S(O)R⁹, —S(O₂)R⁹ or —CN; with the proviso that when Y is ═O,R⁸ cannot be —C(O)R⁹, —C(O)OR¹², —S(O)R⁹, —S(O₂)R⁹ or —CN;

R⁹ is hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, cycloalkylalkyl,aralkyl, heteroaralkyl, heterocyclyl, heterocyclylalkyl, alkenyl,alkynyl or —N(R¹⁰)(R¹¹);

R¹⁰ and R¹¹ are independently selected from the group consisting ofhydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl,heterocyclyl, aralkyl, heteroaralkyl, heterocyclylalkyl, alkenyl andalkynyl;

or R¹⁰ and R¹¹ together with the nitrogen to which they are attached,form a 3-7 membered heterocyclyl ring;

R¹² is alkyl, cycloalkyl, aryl, heteroaryl, cycloalkylalkyl, aralkyl,heteroaralkyl, heterocyclyl, heterocyclylalkyl, alkenyl or alkynyl;

X is O, S, C(R⁵), or NH;

Y is ═O, or (H,H);

m is 1, 2, or 3;

n is 0, 1, 2, or 3;

and

o is 0, 1, 2, or 3;

wherein each alkyl is optionally substituted with 1 to 3 moietiesselected from the group consisting of halo, aryl, cycloalkyl, cyano,hydroxy, alkoxy, alkylthio, amino, —NH(alkyl), —NH(cycloalkyl),—N(alkyl)₂, carboxy and —C(O)O-alkyl; and

wherein each arylene, heteroarylene, heterocyclyl, heterocyclylalkyl,heterocyclylene, cycloalkylene, cycloalkyl, cycloalkylalkyl, aryl,heteroaryl, heterocyclyl, aralkyl, heteroaralkyl, aralkoxy orheteroaralkoxy is optionally substituted with 1 to 4 moieties selectedfrom the group consisting of —CF₃, alkyl, alkenyl, alkynyl, aryl,heteroaryl, aralkyl, alkylaryl, heteroaralkyl, heteroarylalkenyl,heteroarylalkynyl, alkylheteroaryl, hydroxy, hydroxyalkyl, alkoxy,aryloxy, aralkoxy, acyl, aroyl, halo, nitro, cyano, carboxy,alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, alkylsulfonyl,arylsulfonyl, heteroarylsulfonyl, alkylthio, arylthio, heteroarylthio,aralkylthio, heteroaralkylthio, cycloalkyl, heterocyclyl, —C(═N—CN)—NH₂,—C(═NH)—NH₂, —C(═NH)—NH(alkyl), Y₁Y₂NC(O)—, Y₁Y₂NSO₂— and —SO₂NY₁Y₂,wherein Y₁ and Y₂ can be the same or different and are independentlyselected from the group consisting of hydrogen, alkyl, aryl, cycloalkyl,and aralkyl, with the proviso that cycloalkylene and heterocyclylene canbe substituted with ═O.

Compounds represented by formula I are beta-secretase inhibitors usefulfor the prevention and treatment of Alzheimer's disease.

In another aspect, the invention relates to a pharmaceutical compositioncomprising at least one compound of formula I and a pharmaceuticallyacceptable carrier.

In another aspect, the invention comprises a method of inhibiting BACE-1comprising administering at least one compound of formula Ito a patientin need of such treatment. Also claimed is the method of inhibiting theformation, or formation and deposition, of β-amyloid plaques in, on oraround neurological tissue (e.g., the brain) comprising administering atleast one compound of formula Ito a patient in need of such treatment.

More specifically, the invention comprises a method of treating acognitive or neurodegenerative disease comprising administering at leastone compound of formula I to a patient in need of such treatment.Further, the invention comprises the method of treating Alzheimer'sdisease comprising administering at least one compound of formula I to apatient in need of such treatment.

In another aspect, the invention comprises the method of treating acognitive or neurodegenerative disease comprising administering to apatient I need of such treatment a combination of at least one compoundof formula I and at least one compound selected from the groupconsisting of β-secretase inhibitors other than those of formula I,HMG-CoA reductase inhibitors, gamma-secretase inhibitors, non-steroidalanti-inflammatory agents, N-methyl-D-aspartate receptor antagonists,cholinesterase inhibitors and anti-amyloid antibodies.

In a final aspect, the invention relates to a kit comprising in separatecontainers in a single package pharmaceutical compositions for use incombination, in which one container comprises at least one compound offormula I in a pharmaceutically acceptable carrier and a secondcontainer comprises at least one β-secretase inhibitor other than thoseof formula I, HMG-CoA reductase inhibitor, gamma-secretase inhibitor,non-steroidal anti-inflammatory agent, N-methyl-D-aspartate receptorantagonist, cholinesterase inhibitor and/or anti-amyloid antibody in apharmaceutically acceptable carrier, the combined quantities being aneffective amount to treat a cognitive disease or neurodegenerativedisease such as Alzheimer's disease.

DETAILED DESCRIPTION

Referring to formula I, above, preferred compounds of the invention arethose with the following stereochemistry:

In preferred compounds of formula I, R¹ is

R² is preferably is —N(R⁵)C(O)R⁴—, wherein R⁴ is preferably arylene andR⁵ is preferably alkyl. More preferably, R⁴ is phenylene and R⁵ ispropyl. In a preferred embodiment, R⁴ is

Alternatively, R² is heterocyclylene, more preferably R² isheterocyclylene substituted with ═O with the following structure:

R³ is preferably arylene, more preferably, R³ is phenylene orhalo-substituted phenylene. Even more preferably, R³ is

Preferably, m is 2 and n is 1.

R⁷ is preferably hydrogen.

R⁸ is preferably aralkyl or —S(O₂)R⁹ or more preferably R⁸ is

or

Preferably, X is O and Y is O.

In a preferred embodiment of the compound of formula I,

R¹ is

R² is —N(R⁵)C(O)R⁴— or heterocyclylene;

R³ is arylene;

R⁴ is arylene or heterocyclylene;

R⁵ is alkyl;

R⁷ is hydrogen;

R⁸ is aralkyl or —S(O₂)R⁹;

m is 2;

n is 1;

X is O;

and

Y is O.

In the above-preferred embodiment, R³ is preferably phenylene orhalo-substituted phenylene. Specifically, R³ is

In the above-preferred embodiment, R⁴ is preferably arylene,specifically, R⁴ is

Alternatively, R² is heterocyclylene, more preferably R² isheterocyclylene substituted with ═O with the following structure:

In the above-preferred embodiment, R⁸ is

Except where stated otherwise, the following definitions applythroughout the present specification and claims. These definitions applyregardless of whether a term is used by itself or in combination withother terms. Hence the definition of “alkyl” applies to “alkyl” as wellas to the “alkyl” portions of “alkoxy”, “cycloalkyl” and so forth.

As used above, and throughout the specification, the following terms,unless otherwise indicated, shall be understood to have the followingmeanings:

“Patient” includes both human and animals.

“Mammal” means humans and other mammalian animals.

“Alkyl” means an aliphatic hydrocarbon group which may be straight orbranched and comprising about 1 to about 20 carbon atoms in the chain.Preferred alkyl groups contain about 1 to about 12 carbon atoms in thechain. More preferred alkyl groups contain about 1 to about 6 carbonatoms in the chain. Branched means that one or more lower alkyl groupssuch as methyl, ethyl or propyl, are attached to a linear alkyl chain.“Lower alkyl” means a group having about 1 to about 6 carbon atoms inthe chain which may be straight or branched.

“Alkylene” means a difunctional group obtained by removal of a hydrogenatom from an alkyl group that is defined above. Non-limiting examples ofalkylene include methylene and ethylene.

“Alkenyl” means an aliphatic hydrocarbon group containing at least onecarbon-carbon double bond and which may be straight or branched andcomprising about 2 to about 15 carbon atoms in the chain. Preferredalkenyl groups have about 2 to about 12 carbon atoms in the chain; andmore preferably about 2 to about 6 carbon atoms in the chain. Branchedmeans that one or more lower alkyl groups such as methyl, ethyl orpropyl, are attached to a linear alkenyl chain. “Lower alkenyl” meansabout 2 to about 6 carbon atoms in the chain which may be straight orbranched. The term “substituted alkenyl” means that the alkenyl groupmay be substituted by one or more substituents which may be the same ordifferent, each substituent being independently selected from the groupconsisting of halo, alkyl. aryl, cycloalkyl, cyano, alkoxy and—S(alkyl). Non-limiting examples of suitable alkenyl groups includeethenyl, propenyl, n-butenyl, 3-methylbut-2-enyl, n-pentenyl, octenyland decenyl.

“Alkynyl” means an aliphatic hydrocarbon group containing at least onecarbon-carbon triple bond and which may be straight or branched andcomprising about 2 to about 15 carbon atoms in the chain. Preferredalkynyl groups have about 2 to about 12 carbon atoms in the chain; andmore preferably about 2 to about 4 carbon atoms in the chain. Branchedmeans that one or more lower alkyl groups such as methyl, ethyl orpropyl, are attached to a linear alkynyl chain. “Lower alkynyl” meansabout 2 to about 6 carbon atoms in the chain which may be straight orbranched. Non-limiting examples of suitable alkynyl groups includeethynyl, propynyl, 2-butynyl and 3-methylbutynyl. The term “substitutedalkynyl” means that the alkynyl group may be substituted by one or moresubstituents which may be the same or different, each substituent beingindependently selected from the group consisting of alkyl, aryl andcycloalkyl.

“Aryl” means an aromatic monocyclic or multicyclic ring systemcomprising about 6 to about 14 carbon atoms, preferably about 6 to about10 carbon atoms. The aryl group can be optionally substituted with oneor more “ring system substituents” which may be the same or different,and are as defined herein. Non-limiting examples of suitable aryl groupsinclude phenyl and naphthyl.

“Arylene” means a difunctional group obtained by removal of a hydrogenatom from an aryl group that is defined above. Non-limiting examples ofarylene include phenylene and naphthylene.

“Heteroaryl” means an aromatic monocyclic or multicyclic ring systemcomprising about 5 to about 14 ring atoms, preferably about 5 to about10 ring atoms, in which one or more of the ring atoms is an elementother than carbon, for example nitrogen, oxygen or sulfur, alone or incombination. Preferred heteroaryls contain about 5 to about 6 ringatoms. The “heteroaryl” can be optionally substituted by one or more“ring system substituents” which may be the same or different, and areas defined herein. The prefix aza, oxa or thia before the heteroarylroot name means that at least a nitrogen, oxygen or sulfur atomrespectively, is present as a ring atom. A nitrogen atom of a heteroarylcan be optionally oxidized to the corresponding N-oxide. Non-limitingexamples of suitable heteroaryls include pyridyl, pyrazinyl, furanyl,thienyl, pyrimidinyl, pyridone (including N-substituted pyridones),isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazanyl,pyrrolyl, pyrazolyl, triazolyl, 1,2,4-thiadiazolyl, pyrazinyl,pyridazinyl, quinoxalinyl, phthalazinyl, oxindolyl,imidazo[1,2-a]pyridinyl, imidazo[2,1-b]thiazolyl, benzofurazanyl,indolyl, azaindolyl, benzimidazolyl, benzothienyl, quinolinyl,imidazolyl, thienopyridyl, quinazolinyl, thienopyrimidyl,pyrrolopyridyl, imidazopyridyl, isoquinolinyl, benzoazaindolyl,1,2,4-triazinyl, benzothiazolyl and the like. The term “heteroaryl” alsorefers to partially saturated heteroaryl moieties such as, for example,tetrahydroisoquinolyl, tetrahydroquinolyl and the like.

“Heteroarylene” means a difunctional group obtained by removal of ahydrogen atom from a heteroaryl group that is defined above.Non-limiting examples of pyridylene, pyrazinylene, furanylene,thienylene and pyrimidinylene.

“Aralkyl” or “arylalkyl” means an aryl-alkyl-group in which the aryl andalkyl are as previously described. Preferred aralkyls comprise a loweralkyl group. Non-limiting examples of suitable aralkyl groups includebenzyl, 2-phenethyl and naphthalenylmethyl. The bond to the parentmoiety is through the alkyl.

“Alkylaryl” means an alkyl-aryl-group in which the alkyl and aryl are aspreviously described. Preferred alkylaryls comprise a lower alkyl group.Non-limiting example of a suitable alkylaryl group is tolyl. The bond tothe parent moiety is through the aryl.

“Cycloalkyl” means a non-aromatic mono- or multicyclic ring systemcomprising about 3 to about 10 carbon atoms, preferably about 5 to about10 carbon atoms. Preferred cycloalkyl rings contain about 5 to about 7ring atoms. The cycloalkyl can be optionally substituted with one ormore “ring system substituents” which may be the same or different, andare as defined above. Non-limiting examples of suitable monocycliccycloalkyls include cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyland the like. Non-limiting examples of suitable multicyclic cycloalkylsinclude 1-decalinyl, norbornyl, adamantyl and the like, as well aspartially saturated species such as, for example, indanyl,tetrahydronaphthyl and the like.

“Cycloalkylene” means a difunctional group obtained by removal of ahydrogen atom from a cycloalkyl group that is defined above.Non-limiting examples of cycloalkylene include cyclobutylene andcyclopropylene.

“Halo” means fluoro, chloro, bromo or iodo. Preferred are fluoro, chloroand bromo.

“Ring system substituent” means a substituent attached to an aromatic ornon-aromatic ring system which, for example, replaces an availablehydrogen on the ring system. Ring system substituents may be the same ordifferent, each being independently selected from the group consistingof —CF₃, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, alkylaryl,heteroaralkyl, heteroarylalkenyl, heteroarylalkynyl, alkylheteroaryl,hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, acyl, aroyl, halo,nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl,aralkoxycarbonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl,alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio,cycloalkyl, heterocyclyl, ═O, —C(═N—CN)—NH₂, —C(═NH)—NH₂,—C(═NH)—NH(alkyl), Y₁Y₂N—, Y₁Y₂N-alkyl-, Y₁Y₂NC(O)—, Y₁Y₂NSO₂— and—SO₂NY₁Y₂, wherein Y₁ and Y₂ can be the same or different and areindependently selected from the group consisting of hydrogen, alkyl,aryl, cycloalkyl, and aralkyl. “Ring system substituent” may also mean asingle moiety which simultaneously replaces two available hydrogens ontwo adjacent carbon atoms (one H on each carbon) on a ring system.Examples of such moieties are methylene dioxy, ethylenedioxy, —C(CH₃)₂—and the like which form moieties such as, for example:

“Heterocyclyl” means a non-aromatic saturated monocyclic or multicyclicring system comprising about 3 to about 10 ring atoms, preferably about4 to about 7 ring atoms, in which one or more of the atoms in the ringsystem is an element other than carbon, for example nitrogen, oxygen orsulfur, alone or in combination. There are no adjacent oxygen and/orsulfur atoms present in the ring system. Preferred heterocyclyls containabout 5 to about 6 ring atoms. The prefix aza, oxa or thia before theheterocyclyl root name means that at least a nitrogen, oxygen or sulfuratom respectively is present as a ring atom. Any —NH in a heterocyclylring may exist protected such as, for example, as an —N(Boc), —N(CBz),—N(Tos) group and the like; such protections are also considered part ofthis invention. The heterocyclyl can be optionally substituted by one ormore “ring system substituents” which may be the same or different, andare as defined herein. The nitrogen or sulfur atom of the heterocyclylcan be optionally oxidized to the corresponding N-oxide, S-oxide orS,S-dioxide. Non-limiting examples of suitable monocyclic heterocyclylrings include piperidyl, pyrrolidinyl, piperazinyl, morpholinyl,thiomorpholinyl, thiazolidinyl, 1,4-dioxanyl, tetrahydrofuranyl,tetrahydrothiophenyl, lactam, lactone, and the like.

“Heterocyclylene” means a difunctional group obtained by removal of ahydrogen atom from an heterocyclyl group that is defined above.Non-limiting examples of heterocyclylene include piperidylene,pyrrolidinylene, piperazinylene, morpholinylene, thiomorpholinylene,thiazolidinylene, 1,4-dioxanylene, tetrahydrofuranylene andtetrahydrothiophenylene.

It should be noted that in hetero-atom containing ring systems of thisinvention, there are no hydroxyl groups on carbon atoms adjacent to a N,O or S, nor is there an N or S group on a carbon adjacent to anotherheteroatom. Thus, for example, in the ring:

there is no —OH attached directly to carbons marked 2 and 5.

It should also be noted that tautomeric forms such as, for example, themoieties:

are considered equivalent in certain embodiments of this invention.

“Alkynylalkyl” means an alkynyl-alkyl-group in which the alkynyl andalkyl are as previously described. Preferred alkynylalkyls contain alower alkynyl and a lower alkyl group. The bond to the parent moiety isthrough the alkyl. Non-limiting examples of suitable alkynylalkyl groupsinclude propargylmethyl.

“Heteroaralkyl” means a heteroaryl-alkyl-group in which the heteroaryland alkyl are as previously described. Preferred heteroaralkyls containa lower alkyl group. Non-limiting examples of suitable heteroaralkylgroups include pyridylmethyl, and quinolin-3-ylmethyl. The bond to theparent moiety is through the alkyl.

“Heteroaralkylthio” means a heteroaralkyl-S— group in which theheteroaralkyl is as previously described. Preferred heteroaralkylthioscontain a lower alkyl group. The bond to the parent moiety is throughthe sulfur.

“Heteroarylalkenyl” means a heteroaryl-alkenyl group in which theheteroaryl and the alkenyl are as previously described. Preferredheteroarylalkenyls contain a lower alkenyl group. The bond to the parentmoiety is through the alkenyl.

“Heteroarylalkynyl” means a heteroaryl-alkynyl group in which theheteroaryl and the alkynyl are as previously described. Preferredheteroarylalkynyls contain a lower alkynyl group. The bond to the parentmoiety is through the alkynyl.

“Hydroxyalkyl” means a HO-alkyl-group in which alkyl is as previouslydefined. Preferred hydroxyalkyls contain lower alkyl. Non-limitingexamples of suitable hydroxyalkyl groups include hydroxymethyl and2-hydroxyethyl.

“Acyl” means an H—C(O)—, alkyl-C(O)— or cycloalkyl-C(O)—, group in whichthe various groups are as previously described. The bond to the parentmoiety is through the carbonyl. Preferred acyls contain a lower alkyl.Non-limiting examples of suitable acyl groups include formyl, acetyl andpropanoyl.

“Aroyl” means an aryl-C(O)— group in which the aryl group is aspreviously described. The bond to the parent moiety is through thecarbonyl. Non-limiting examples of suitable groups include benzoyl and1-naphthoyl.

“Alkoxy” means an alkyl-O— group in which the alkyl group is aspreviously described. Non-limiting examples of suitable alkoxy groupsinclude methoxy, ethoxy, n-propoxy, isopropoxy and n-butoxy. The bond tothe parent moiety is through the ether oxygen.

“Alkoxyalkyl” means an alkoxy-alkyl group in which the alkoxy and alkylgroups are as previously described. Non-limiting examples of suitablealkoxyalkyl groups include ethoxyethyl, methoxymethyl and ethoxymethyl.The bond to the parent moiety is through the alkyl group.

“Aryloxy” means an aryl-O— group in which the aryl group is aspreviously described. Non-limiting examples of suitable aryloxy groupsinclude phenoxy and naphthoxy. The bond to the parent moiety is throughthe ether oxygen.

“Aralkoxy” means an aralkyl-O— group in which the aralkyl group is aspreviously described. Non-limiting examples of suitable aralkyloxygroups include benzyloxy and 1- or 2-naphthalenemethoxy. The bond to theparent moiety is through the ether oxygen.

“Alkylheteroaryl” means an alkyl-heteroaryl group in which the alkyl andheteroaryl groups are as previously described. The bond to the parentmoiety is through the heteroaryl.

“Alkylthio” means an alkyl-S— group in which the alkyl group is aspreviously described. Non-limiting examples of suitable alkylthio groupsinclude methylthio and ethylthio. The bond to the parent moiety isthrough the sulfur.

“Arylthio” means an aryl-S— group in which the aryl group is aspreviously described. Non-limiting examples of suitable arylthio groupsinclude phenylthio and naphthylthio. The bond to the parent moiety isthrough the sulfur.

“Aralkylthio” means an aralkyl-S— group in which the aralkyl group is aspreviously described. Non-limiting example of a suitable aralkylthiogroup is benzylthio. The bond to the parent moiety is through thesulfur.

“Alkoxycarbonyl” means an alkyl-O—C(O)— group in which the alkyl groupis as previously described. Non-limiting examples of suitablealkoxycarbonyl groups include methoxycarbonyl and ethoxycarbonyl. Thebond to the parent moiety is through the carbonyl.

“Aryloxycarbonyl” means an aryl-O—C(O)— group in which the aryl group isas previously described. Non-limiting examples of suitablearyloxycarbonyl groups include phenoxycarbonyl and naphthoxycarbonyl.The bond to the parent moiety is through the carbonyl.

“Aralkoxycarbonyl” means an aralkyl-O—C(O)— group in which the aralkylgroup is as previously described. Non-limiting example of a suitablearalkoxycarbonyl group is benzyloxycarbonyl. The bond to the parentmoiety is through the carbonyl.

“Alkylsulfonyl” means an alkyl-S(O₂)— group in which the alkyl group isas previously described. Preferred groups are those in which the alkylgroup is lower alkyl. The bond to the parent moiety is through thesulfonyl.

“Arylsulfonyl” means an aryl-S(O₂)— group in which the aryl group is aspreviously described. The bond to the parent moiety is through thesulfonyl.

“Cycloalkylalkyl” means a cycloalkyl-alkyl-group in which the cycloalkyland alkyl group is as previously described. Preferred groups are thosein which the alkyl group is lower alkyl. The bond to the parent moietyis through the alkyl.

“Heteroaralkoxy” means an heteroaralkyl-O— group in which theheteroaralkyl group is as previously described. The bond to the parentmoiety is through the ether oxygen.

“Heteroarylsulfonyl” means a heteroaryl-S(O₂)— group in which theheteroaryl group is as previously described. The bond to the parentmoiety is through the sulfonyl.

“Heteroarylthio” means a heteroaryl-S— group in which the heteroarylgroup is as previously described. The bond to the parent moiety isthrough the sulfur.

“Heterocyclylalkyl” means a heterocyclyl-alkyl group in which theheterocyclyl and the alkyl are as previously described. The bond to theparent moiety is through the alkyl.

The term “substituted” means that one or more hydrogens on thedesignated atom is replaced with a selection from the indicated group,provided that the designated atom's normal valency under the existingcircumstances is not exceeded, and that the substitution results in astable compound. Combinations of substituents and/or variables arepermissible only if such combinations result in stable compounds. By“stable compound” or “stable structure” is meant a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and formulation into an efficacious therapeuticagent.

The term “optionally substituted” means optional substitution with thespecified groups, radicals or moieties.

With reference to the number of moieties (e.g., substituents, groups orrings) in a compound, unless otherwise defined, the phrases “one ormore” and “at least one” mean that there can be as many moieties aschemically permitted, and the determination of the maximum number ofsuch moieties is well within the knowledge of those skilled in the art.With respect to the compositions and methods comprising the use of “atleast one compound of formula I,” one to three compounds of formula Ican be administered at the same time, preferably one.

The wavy line

as a bond generally indicates a mixture of, or either of, the possibleisomers, e.g., containing (R)- and (S)-stereochemistry. For example,

means containing both

Lines drawn into the ring systems, such as, for example:

indicate that the indicated line (bond) may be attached to any of thesubstitutable ring carbon atoms.

As well known in the art, a bond drawn from a particular atom wherein nomoiety is depicted at the terminal end of the bond indicates a methylgroup bound through that bond to the atom, unless stated otherwise. Forexample:

The term “isolated” or “in isolated form” for a compound refers to thephysical state of said compound after being isolated from a syntheticprocess or natural source or combination thereof. The term “purified” or“in purified form” for a compound refers to the physical state of saidcompound after being obtained from a purification process or processesdescribed herein or well known to the skilled artisan, in sufficientpurity to be characterizable by standard analytical techniques describedherein or well known to the skilled artisan.

It should also be noted that any heteroatom with unsatisfied valences inthe text, schemes, examples and Tables herein is assumed to have thehydrogen atom(s) to satisfy the valences.

When a functional group in a compound is termed “protected”, this meansthat the group is in modified form to preclude undesired side reactionsat the protected site when the compound is subjected to a reaction.Suitable protecting groups will be recognized by those with ordinaryskill in the art as well as by reference to standard textbooks such as,for example, T. W. Greene et al, Protective Groups in organic Synthesis(1991), Wiley, N.Y.

When any variable (e.g., aryl, heterocycle, R², etc.) occurs more thanone time in any constituent or in Formula I, its definition on eachoccurrence is independent of its definition at every other occurrence.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombination of the specified ingredients in the specified amounts.

Prodrugs and solvates of the compounds of the invention are alsocontemplated herein. The term “prodrug”, as employed herein, denotes acompound that is a drug precursor which, upon administration to asubject, undergoes chemical conversion by metabolic or chemicalprocesses to yield a compound of Formula I or a salt and/or solvatethereof. A discussion of prodrugs is provided in T. Higuchi and V.Stella, Pro-drugs as Novel Delivery Systems (1987) 14 of the A.C.S.Symposium Series, and in Bioreversible Carriers in Drug Design, (1987)Edward B. Roche, ed., American Pharmaceutical Association and PergamonPress, both of which are incorporated herein by reference thereto.

“Solvate” means a physical association of a compound of this inventionwith one or more solvent molecules. This physical association involvesvarying degrees of ionic and covalent bonding, including hydrogenbonding. In certain instances the solvate will be capable of isolation,for example when one or more solvent molecules are incorporated in thecrystal lattice of the crystalline solid. “Solvate” encompasses bothsolution-phase and isolatable solvates. Non-limiting examples ofsuitable solvates include ethanolates, methanolates, and the like.“Hydrate” is a solvate wherein the solvent molecule is H₂O.

The compounds of Formula I can form salts which are also within thescope of this invention. Reference to a compound of Formula I herein isunderstood to include reference to salts thereof, unless otherwiseindicated. The term “salt(s)”, as employed herein, denotes acidic saltsformed with inorganic and/or organic acids, as well as basic saltsformed with inorganic and/or organic bases. In addition, when a compoundof Formula I contains both a basic moiety, such as, but not limited to apyridine or imidazole, and an acidic moiety, such as, but not limited toa carboxylic acid, zwitterions (“inner salts”) may be formed and areincluded within the term “salt(s)” as used herein. Pharmaceuticallyacceptable (i.e., non-toxic, physiologically acceptable) salts arepreferred, although other salts are also useful. Salts of the compoundsof the Formula I may be formed, for example, by reacting a compound ofFormula I with an amount of acid or base, such as an equivalent amount,in a medium such as one in which the salt precipitates or in an aqueousmedium followed by lyophilization.

Exemplary acid addition salts include acetates, ascorbates, benzoates,benzenesulfonates, bisulfates, borates, butyrates, citrates,camphorates, camphorsulfonates, fumarates, hydrochlorides,hydrobromides, hydroiodides, lactates, maleates, methanesulfonates,naphthalenesulfonates, nitrates, oxalates, phosphates, propionates,salicylates, succinates, sulfates, tartarates, thiocyanates,toluenesulfonates (also known as tosylates,) and the like. Additionally,acids which are generally considered suitable for the formation ofpharmaceutically useful salts from basic pharmaceutical compounds arediscussed, for example, by P. Stahl et al, Camille G. (eds.) Handbook ofPharmaceutical Salts. Properties, Selection and Use. (2002) Zurich:Wiley-VCH; S. Berge et al, Journal of Pharmaceutical Sciences (1977)66(1) 1-19; P. Gould, International J. of Pharmaceutics (1986) 33201-217; Anderson et al, The Practice of Medicinal Chemistry (1996),Academic Press, New York; and in The Orange Book (Food & DrugAdministration, Washington, D.C. on their website). These disclosuresare incorporated herein by reference thereto.

Exemplary basic salts include ammonium salts, alkali metal salts such assodium, lithium, and potassium salts, alkaline earth metal salts such ascalcium and magnesium salts, salts with organic bases (for example,organic amines) such as dicyclohexylamines, t-butyl amines, and saltswith amino acids such as arginine, lysine and the like. Basicnitrogen-containing groups may be quarternized with agents such as loweralkyl halides (e.g. methyl, ethyl, and butyl chlorides, bromides andiodides), dialkyl sulfates (e.g. dimethyl, diethyl, and dibutylsulfates), long chain halides (e.g. decyl, lauryl, and stearylchlorides, bromides and iodides), aralkyl halides (e.g. benzyl andphenethyl bromides), and others.

All such acid salts and base salts are intended to be pharmaceuticallyacceptable salts within the scope of the invention and all acid and basesalts are considered equivalent to the free forms of the correspondingcompounds for purposes of the invention.

Compounds of Formula I, and salts, solvates and prodrugs thereof, mayexist in their tautomeric form (for example, as an amide or iminoether). All such tautomeric forms are contemplated herein as part of thepresent invention.

All stereoisomers (for example, geometric isomers, optical isomers andthe like) of the present compounds (including those of the salts,solvates and prodrugs of the compounds as well as the salts and solvatesof the prodrugs), such as those which may exist due to asymmetriccarbons on various substituents, including enantiomeric forms (which mayexist even in the absence of asymmetric carbons), rotameric forms,atropisomers, and diastereomeric forms, are contemplated within thescope of this invention, as are positional isomers (such as, forexample, 4-pyridyl and 3-pyridyl). Individual stereoisomers of thecompounds of the invention may, for example, be substantially free ofother isomers, or may be admixed, for example, as racemates or with allother, or other selected, stereoisomers. The chiral centers of thepresent invention can have the S or R configuration as defined by theIUPAC 1974 Recommendations. The use of the terms “salt”, “solvate”“prodrug” and the like, is intended to equally apply to the salt,solvate and prodrug of enantiomers, stereoisomers, rotamers, tautomers,positional isomers, racemates or prodrugs of the inventive compounds.

Compounds represented by formula I are beta-secretase inhibitors usefulfor the prevention and treatment of Alzheimer's disease.

An aspect of this invention is a method of treating a mammal (e.g.,human) having a disease or condition mediated or exacerbated by BACE-1(an aspartyl protease) by administering a therapeutically effectiveamount of at least one compound of Formula I, or a pharmaceuticallyacceptable salt or solvate of said compound to the mammal.

“Effective amount” or “therapeutically effective amount” is meant todescribe an amount of compound or a composition of the present inventioneffective in inhibiting BACE-1 and thus producing the desiredtherapeutic effect in a suitable patient.

A preferred dosage is about 0.001 to 1000 mg/kg of body weight/day ofthe compound of Formula I or a pharmaceutically acceptable salt orsolvate thereof. An especially preferred dosage is about 0.01 to 30mg/kg of body weight/day of a compound of Formula I, or apharmaceutically acceptable salt or solvate of said compound.

Still yet another aspect of this invention is a method of treating acognitive or neurodegenerative disease, such as Alzheimer's disease,comprising administering to a mammal in need of such treatment atherapeutically effective amount of at least one compound of Formula I,or a pharmaceutically acceptable salt or solvate of said compound.

A further aspect of this invention is a method for treating a cognitiveor neurodegenerative disease such as Alzheimer's disease, comprisingadministering to a mammal a therapeutically effective amount of at leastone compound of Formula I, or a pharmaceutically acceptable salt orsolvate of said compound.

This invention is also directed to pharmaceutical compositions, whichcomprise at least one compound of Formula I, or a pharmaceuticallyacceptable salt or solvate of said compound and at least onepharmaceutically acceptable carrier.

This invention is also directed to pharmaceutical compositions for thetreatment of neurodegenerative diseases such as Alzheimer's diseasewhich comprise an effective treating amount of at least one compound ofFormula I, or a pharmaceutically acceptable salt or solvate of saidcompound and at least one pharmaceutically acceptable carrier.

For the combination aspect, the use of any β-secretase inhibitor otherthan those of formula I is contemplated; β-secretase inhibitory activitycan be determined by the procedures described below. Useful β-secretaseinhibitors are those disclosed in, but are not limited to, WO 02/02505,WO 02/02506, WO 02/02512, WO 02/02518, WO 02/02520 and WO 02/088101.

Still yet other aspects of this invention are combinations of a compoundof Formula I, or a pharmaceutically acceptable salt or solvate of saidcompound and other compounds as described below.

Accordingly, included within the invention is a method for treatingneurodegenerative diseases such as Alzheimer's, comprising administeringto a mammal (e.g., a female or male human)

a. an amount of a first compound, said first compound being a compoundof Formula I, or a pharmaceutically acceptable salt or solvate of saidcompound; and

b. an amount of a second compound, said second compound being acholinesterase inhibitor.

Cholinesterase inhibitors for use in the combination include acetyl-and/or butyrylchiolinesterase inhibitors. Examples of cholinesteraseinhibitors include tacrine, donepezil, rivastigmine, galantamine,pyridostigmine and neostigmine.

Accordingly, included within the invention is a method for treatingneurodegenerative diseases such as Alzheimer's, comprising administeringto a mammal (e.g., a female or male human)

a. an amount of a first compound, said first compound being a compoundof Formula I, or a pharmaceutically acceptable salt or solvate of saidcompound; and

b. an amount of a second compound, said second compound being ananti-amyloid antibody. Anti amyloid antibodies are described, forexample, in Hock et al, Nature Medicine, 8 (2002), p. 1270-1275.

Accordingly, included within the invention is a method for treatingneurodegenerative diseases such as Alzheimer's, comprising administeringto a mammal (e.g., a female or male human)

a. an amount of a first compound, said first compound being a compoundof Formula I, or a pharmaceutically acceptable salt or solvate of saidcompound; and

b. an amount of a second compound, said second compound being ananti-inflammatory compound. Examples of anti-inflammatory compoundsinclude but are non limited to non-steroidal anti-inflammatory drugssuch as diclofenac (Voltaren, Cataflam), diflunisal (Dolobid), etodolac(Lodine), flurbiprofen (Ansaid), ibuprofen (Motrin, Advil), indomethacin(Indocin), ketoprofen (Orudis, Oruvail), ketorolac (Toradol), nabumetone(Relafen), naproxen (Naprosyn, Alleve), oxaprozin (Daypro), piroxicam(Feldene), sulindac (Clinoril), tolmetin (Tolectin), celecoxib(Celebrex) and rofecoxib (Vioxx).

Accordingly, included within the invention is a method for treatingneurodegenerative diseases such as Alzheimer's, comprising administeringto a mammal (e.g., a female or male human)

a. an amount of a first compound, said first compound being a compoundof Formula I, or a pharmaceutically acceptable salt or solvate of saidcompound; and

b. an amount of a second compound, said second compound being a gammasecretase inhibitor. Gamma-secretase inhibitors for use in thecombination of this invention can be determined by procedures known inthe art. Typical gamma-secretase inhibitors include, but are not limitedto, those described in WO 03/013527, U.S. Pat. No. 6,683,091, WO03/066592, U.S. Ser. No. 10/663,042, filed Sep. 16, 2003, WO 00/247671,WO 00/050391, WO 00/007995 and WO 03/018543.

Accordingly, included within the invention is a method for treatingneurodegenerative diseases such as Alzheimer's, comprising administeringto a mammal (e.g., a female or male human)

a. an amount of a first compound, said first compound being a compoundof Formula I, or a pharmaceutically acceptable salt or solvate of saidcompound; and

b. an amount of a second compound, said second compound being a HMG-CoAreductase inhibitor compound. HMG-CoA reductase inhibitors for use incombination with compounds of formula I include the “stains,” e.g.,atorvastatin, lovastatin, simvistatin, pravastatin, fluvastatin androsuvastatin.

Accordingly, included within the invention is a method for treatingneurodegenerative diseases such as Alzheimer's, comprising administeringto a mammal (e.g., a female or male human)

a. an amount of a first compound, said first compound being a compoundof Formula I, or a pharmaceutically acceptable salt or solvate of saidcompound; and

b. an amount of a second compound, said second compound being aN-methyl-D-aspartate receptor antagonist. A suitableN-methyl-D-aspartate receptor antagonist is, for example, memantine.

Preferably, the pharmaceutical preparation is in a unit dosage form. Insuch form, the preparation is subdivided into suitably sized unit dosescontaining appropriate quantities of the active component, e.g., aneffective amount to achieve the desired purpose.

The quantity of active compound in a unit dose of preparation may bevaried or adjusted from about 1 mg to about 1000 mg, preferably fromabout 1 mg to about 50 mg, more preferably from about 1 mg to about 25mg, according to the particular application.

The actual dosage employed may be varied depending upon the requirementsof the patient and the severity of the condition being treated.Determination of the proper dosage regimen for a particular situation iswithin the skill of the art. For convenience, the total daily dosage maybe divided and administered in portions during the day as required.

The amount and frequency of administration of the compounds of theinvention and/or the pharmaceutically acceptable salts thereof will beregulated according to the judgment of the attending clinicianconsidering such factors as age, condition and size of the patient aswell as severity of the symptoms being treated. A typical recommendeddaily dosage regimen for oral administration can range from about 1mg/day to about 300 mg/day, preferably 1 mg/day to 50 mg/day, in two tofour divided doses.

The amount and frequency of administration of the compounds of thecombinations (beta secreatse inhibitors other than those of formula I,NSAIDS, statin drugs, cholinesterase inhibitors, etc.), and/or thepharmaceutically acceptable salts thereof will be regulated according tothe judgment of the attending clinician considering such factors as age,condition and size of the patient as well as severity of the symptomsbeing treated.

For preparing pharmaceutical compositions from the compounds describedby this invention, inert, pharmaceutically acceptable carriers can beeither solid or liquid. Solid form preparations include powders,tablets, dispersible granules, capsules, cachets and suppositories. Thepowders and tablets may be comprised of from about 5 to about 95 percentactive ingredient. Suitable solid carriers are known in the art, e.g.magnesium carbonate, magnesium stearate, talc, sugar or lactose.Tablets, powders, cachets and capsules can be used as solid dosage formssuitable for oral administration. Examples of pharmaceuticallyacceptable carriers and methods of manufacture for various compositionsmay be found in A. Gennaro (ed.), Remington's Pharmaceutical Sciences,18th Edition, (1990), Mack Publishing Co., Easton, Pa.

For preparing suppositories, a low melting wax such as a mixture offatty acid glycerides or cocoa butter is first melted, and the activeingredient is dispersed homogeneously therein as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool and thereby solidify.

Liquid form preparations include solutions, suspensions and emulsions.As an example may be mentioned water or water-propylene glycol solutionsfor parenteral injection. Liquid form preparations may also includesolutions for intranasal administration.

Aerosol preparations suitable for inhalation may include solutions andsolids in powder form, which may be in combination with apharmaceutically acceptable carrier, such as an inert compressed gas.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for eitheroral or parenteral administration. Such liquid forms include solutions,suspensions and emulsions.

The compounds of the invention may also be deliverable transdermally.The transdermal compositions can take the form of creams, lotions,aerosols and/or emulsions and can be included in a transdermal patch ofthe matrix or reservoir type as are conventional in the art for thispurpose.

Preferably the compound is administered orally.

Preferably, the pharmaceutical preparation is in unit dosage form. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component, e.g., an effectiveamount to achieve the desired purpose.

When a compound of formula I is used in combination with β-secretaseinhibitors other than those of formula I, an HMG-CoA reductaseinhibitor, a gamma-secretase inhibitor, a non-steroidalanti-inflammatory agent, an N-methyl-D-aspartate receptor antagonist, acholinesterase inhibitor or an anti-amyloid antibody to treat acognitive disorder or neurodegenerative disorder, the active componentsmay be co-administered simultaneously or sequentially, or a singlepharmaceutical composition comprising a compound of formula I and one ofthe other agents in a pharmaceutically acceptable carrier can beadministered. The components of the combination can be administeredindividually or together in any conventional oral or parenteral dosageform such as capsule, tablet, powder, cachet, suspension, solution,suppository, nasal spray, etc. The dosage of the β-secretase inhibitorsother than those of formula I, HMG-CoA reductase inhibitor,gamma-secretase inhibitor, non-steroidal anti-inflammatory agent,N-methyl-D-aspartate receptor antagonist, cholinesterase inhibitor oranti-amyloid antibody can be determined from published material, and mayrange from 0.001 to 100 mg/kg body weight.

When separate pharmaceutical compositions of a compound of formula I anda β-secretase inhibitors other than those of formula I, an HMG-CoAreductase inhibitor, a gamma-secretase inhibitor, a non-steroidalanti-inflammatory agent, an N-methyl-D-aspartate receptor antagonist, acholinesterase inhibitor or an anti-amyloid antibody are to beadministered, they can be provided in a kit comprising in a singlepackage, one container comprising a compound of formula I in apharmaceutically acceptable carrier, and a separate container comprisingthe other agent in a pharmaceutically acceptable carrier, with thecompound of formula I and the other agent being present in amounts suchthat the combination is therapeutically effective. A kit is advantageousfor administering a combination when, for example, the components mustbe administered at different time intervals or when they are indifferent dosage forms.

The invention also includes multi-agent compositions, kits and methodsof treatment, e.g., a compound of formula I can be administered incombination with an HMG-CoA reductase inhibitor and a non-steroidalanti-inflammatory agent

Compounds of Formula I can be produced by processes known to thoseskilled in the art using either solution phase or solid phase synthesisas shown in the following reaction schemes, in the preparations andexamples below, but those skilled in the art will recognize that otherprocedures can also be suitable.

In the Schemes and in the Examples below, the following abbreviationsare used:

methyl: Me; ethyl: Et; propyl: Pr; butyl: Bu; benzyl: Bn

ethyl acetate: EtOAc

benzyloxycarbonyl: Cbz

N,N-dimethylformamide: DMF:

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: EDC or EDCI

room temperature: RT

hour: h

minute: min

retention time: t_(R)

trifluoroacetic acid: TFA

tetrahydrofuran: THF

1-hydroxybenzotriazole: HOBt

methanol: MeOH

ethanol: EtOH

acetic acid: AcOH

dimethylsulfoxide: DMSO

lithium diisopropylamide: LDA

tert-dimethylsilyl chloride: TBSCl

tert-dimethylsilyl: TBS

triphenyl phosphine: PPh₃

diisopropyl azodicarboxylate: DIAD

copper(I) bromide-dimethyl sulfide: CuBr-Me₂S

tertiary butyloxycarbonyl: Boc

Palladium tetrakis (Triphenylphosphine): Pd(PPh₃)₄

Triphenylphosphine: PPh₃

Tetrabutylammonium fluoride: TBAF

Triethylamine: Et₃N, NEt₃ or TEA

Lithium borohydride: LiBH₄

benzyl bromide: BnBr

Di-tert-butyl dicarbonate: (Boc)₂O

4-dimethylaminopyridine: DMAP

butyllithium: BuLi

Benzyl chloride: BnCl

oxalyl chloride: (COCl)₂

Preparative thin layer chromatography: PTLC

thin layer chromatography: TLC

nuclear magnetic resonance: NMR

liquid chromatography mass spectrometry: LCMS

Diisopropylamine: DIPA

dimethylacetamide: DMA

pivaloyl chloride; PivCl.

PREPARATIVE EXAMPLE 1

To 4-chloro-1-butanol (49.5 g, 0.456 mmol) in an ice-water bath wasadded propylamine (150 ml, 1.82 mol). The mixture was slowly warmed toRT and stirred for 64 h. Then the mixture was refluxed for 5 h andevaporated under reduced pressure. The residue was partitioned betweenether (3×250 ml) and 40% aqueous NaOH (400 ml). The combined organiclayer was dried (MgSO₄), concentrated, and distilled to give the product(14.87 g, 25%). ¹H-NMR (CDCl₃): δ=3.56 (m, 4H), 2.59 (m, 4H), 1.4-1.8(m, 6H), 0.93 (m, 3H).

A solution of the product of Step 1 (8.655 g, 66.07 mmol), TBSCl (20.12g, 133.5 mmol), imidazole (13.50 g, 198.3 mmol), and catalytic amount ofDMAP in anhydrous CH₂Cl₂ (180 ml) was stirred at RT for 16 h. Themixture was washed with 0.5N NaOH (100 ml), dried (Na₂SO₄),concentrated, and taken up in DMF (20 ml). To the resulting solutionwere added HOBt (10.73 g, 79.45 mmol), EDCI (15.38 g, 80.25 mmol),Triethylamine (Et₃N, NEt₃ or TEA) (29.0 ml, 208 mmol), and mono-methylisophthalate (10.23 g, 56.78 mmol). The mixture was stirred at RT for 3days and evaporated to dryness. The residue was partitioned betweenCH₂Cl₂ (200 ml) and 0.5N NaOH (200 ml). The organic layer was washedwith aqueous NH₄Cl (100 ml), dried (MgSO₄), and purified by columnchromatography (gradient MeOH/CH₂Cl₂ 0-3%) to give the product (8.829 g,38%). MS m/e 408 (M+H)⁺

A mixture of the product of Step 2 (8.829 g, 21.69 mmol) and LiOH—H₂O(1.021 g, 24.33 mmol) in MeOH (75 ml) and water (25 ml) was stirred atRT for 16 h. The mixture was evaporated to dryness and the residue wastaken up in DMF (80 ml). To the resulting solution were addedtriethylamine (3.0 ml, 21 mmol), EDCI (4.233 g, 22.08 mmol), HOBt (2.974g, 22.01 mmol), and L-tyrosine methyl ester (4.235 g, 21.69 mmol). Themixture was stirred at RT for 16 h and concentrated. The residue waspartitioned between CH₂Cl₂ (250 ml) and aqueous NH₄Cl (50 ml). Theorganic layer was washed with 5% sodium bicarbonate (100 ml), dried(MgSO₄), and purified by column chromatography (gradient MeOH/CH₂Cl₂0-3.5%) to give the product (7.84 g, 63%). MS m/e 571 (M+H)⁺

To a solution of the product of Step 3 (3.50 g, 6.14 mmol) in THF (100ml) was added 1M TBAF in THF (9.2 ml) and the mixture was stirred at RTfor 4.5 h. The mixture was concentrated and purified by columnchromatography (gradient MeOH/CH₂Cl₂ 0-4%) to give the product (2.40 g,86%). MS m/e 457 (M+H)⁺

A mixture of the product of Step 4 (1.22 g, 2.68 mmol),tributylphosphine (995 μl, 4.01 mmol), and1,1′-(azodicarbonyl)dipiperidine (1.01 g, 4.01 mmol) in benzene (150 ml)and THF (19 ml) was stirred at RT for 22 h. The mixture was concentratedand the residue was dissolved in EtOAc (200 ml) and washed with 1N HCl(100 ml). The organic layer was extracted with saturated sodiumbicarbonate and brine, dried (MgSO₄), concentrated, and purified bycolumn chromatography (gradient MeOH/CH₂Cl₂ 0-2%) to give the product(0.583 g, 50%). MS m/e 439 (M+H)⁺

To a solution of the product of Step 5 (580 mg, 1.32 mmol) in absoluteEtOH (20 ml) in an ice-water bath was added 2M lithium borohydride inTHF (3.3 ml). The mixture was stirred in the ice-water bath for 10 minthen at RT for 4 h. The reaction was quenched with water (1 ml) and 5%citric acid (5 ml). The mixture was concentrated and extracted withEtOAc (3×50 ml). The combined organic layer was washed with saturatedsodium bicarbonate (20 ml) and brine, dried (MgSO₄), concentrated, andpurified by column chromatography (gradient MeOH/CH₂Cl₂ 0-5%) to givethe product (502 mg, 93%). MS m/e 411 (M+H)⁺

To a solution of piperazinone (10.0 g, 100 mmol), Triethylamine (20.2 g,200 mmol), and DMAP (50 mg) in CH₂Cl₂ (250 ml) in an ice water bath wasadded (Boc)₂O (22.9 g, 105 mmol) slowly. The mixture was stirred in theice-water bath for 1 h and at RT for 4.5 h. The mixture was diluted withCH₂Cl₂ (250 ml), washed with water (200 ml), 5% citric acid (200 ml), 1NHCl (200 ml), saturated sodium bicarbonate (20 ml) and brine. Theorganic layer was dried (MgSO₄) and concentrated to give the product(18.0 g, 90%). MS m/e 201 (M+H)⁺

To a solution of the product of Step 7 (10.0 g, 50.0 mmol) in anhydrousDMF (250 ml) in an ice-water bath were added sodium hydride (2.40 g,60.0 mmol) and benzyl chloride (6.60 g, 52.5 mmol). The mixture wasstirred at RT for 4.5 h. The reaction was quenched with water (10 ml),diluted with CH₂Cl₂ (500 ml), and washed with water (2×250 ml). Theorganic layer was extracted with saturated NH₄ (200 ml), dried (MgSO₄),concentrated, and purified by column chromatography (gradientMeOH/CH₂Cl₂ 0-5%) to give the product (10.7 g, 74%). ¹H-NMR (CDCl₃):δ=7.2-7.3 (m, 5H), 4.57 (s, 2H), 4.10 (s, 2H), 3.53 (m, 2H), 3.19 (m,2H), 1.41 (s, 9H).

A mixture of the product of Step 6 (123 mg, 0.300 mmol) and Dess-Martinperiodinane (256 mg, 0.602 mmol) in CH₂Cl₂ (15 ml) was stirred at RT for30 min. The mixture was diluted with CH₂Cl₂ (50 ml), washed with 1NNa₂S₂O₃ (20 ml) and saturated NaHCO₃, dried (MgSO₄), and concentrated togive the crude aldehyde.

To a solution of the product of Step 8 (261 mg, 0.900 mmol) in anhydrousTHF (5 ml) in a dry ice-acetone bath was added 2M LDA (0.45 ml) and themixture was stirred for 1 h. A solution of the above aldehyde in THF (5ml) was added and the mixture was stirred in the dry ice-acetone bathfor 2 h. The reaction was quenched with saturated NH₄Cl (4 ml), dilutedwith CH₂Cl₂ (50 ml), and washed with water (30 ml). The organic layerwas extracted with saturated NH₄Cl and brine, dried (MgSO₄),concentrated, and purified by PTLC (5% MeOH/CH₂Cl₂) to give the product(100 mg, 48%). MS m/e 699 (M+H)⁺

A solution of the product of Step 9 (100 mg, 0.143 mmol) in 15%TFA/CH₂Cl₂ (10 ml) was stirred at RT for 75 min. The mixture wasconcentrated and purified by PTLC (5% MeOH/CH₂Cl₂) to give:

fraction A (15 mg, 18%). ¹H-NMR (CDCl₃): δ=6.4-7.8 (m, 13H), 5.83 (m,1H), 3.9-5.0 (m, 6H), 2.7-3.8 (m, 11H), 1.5-2.0 (m, 5H), 1.2-1.5 (m,3H), 0.4-1.0 (m, 3H). MS m/e 599 (M+H)⁺

fraction B (18 mg, 21%). ¹H-NMR (CDCl₃): δ=7.0-7.6 (m, 10H), 6.6-7.0 (m,3H), 5.7-6.3 (m, 1H), 3.8-4.8 (m, 6H), 2.6-3.7 (m, 12H), 1.2-2.0 (m,7H), 0.98 (m, 1H), 0.62 (m, 2H). MS m/e 599 (M+H)⁺

PREPARATIVE EXAMPLE 2

n-Bromopropane (24.6 g, 0.200 mol) was added to allylamine (451 g, 0.800mol) in an ice-water bath and the mixture was stirred at RT for 3 d. Themixture was distilled to give a solid, 5 g of which was dissolved in DMF(50 ml). To this solution were added mono-methyl isophthalate (1.86 g,10.0 mmol), HOBt (2.70 g, 20.0 mmol), and EDCI (3.83 g, 20.0 mmol). Themixture was stirred at RT for 16 h and diluted with EtOAc (300 ml) and1N NaOH (100 ml). The organic layer was washed with 1N HCl (100 ml),water (100 ml), saturated sodium bicarbonate (100 ml), and brine (100ml), dried (MgSO₄), and concentrated to give the product (2.20 g, 84%).MS m/e 262 (M+H)⁺

A mixture of the product of Step 1 (2.20 g, 8.42 mmol) in MeOH (25 ml)and 1N HCl (18 ml) was stirred at RT for 18 h. The mixture wasconcentrated and the residue was partitioned between 1N HCl (20 ml) andether (2×100 ml). The combined organic layer was dried (Na₂SO₄) andconcentrated to give the product (2.10 g, 100%). MS m/e 248 (M+H)⁺

To an ice-cold solution of N-Boc-D-serine methyl ester (10.0 g, 45.6mmol) in DMF (150 ml) were added imidazole (9.26 g, 136 mmol) and TBSCI(7.56 g, 50.16 mmol). The mixture was stirred at RT for 20 h andconcentrated. The residue was dissolved with EtOAc (300 ml) andextracted with saturated NH₄Cl and sodium bicarbonate. The organic layerwas dried (MgSO₄) and concentrated to give the product (16.5 g, 100%).MS m/e 356 (M+Na)⁺

To a solution of the product of Step 3 (16.5 g, 45.6 mmol) in THF (150ml) was added 2M lithium borohydride in THF (37.1 ml) slowly. Themixture was stirred at RT for 2.5 h. The reaction was quenched withsaturated NH₄Cl and extracted with EtOAc (2×250 ml). The combinedorganic layer was washed with saturated NH₄Cl (100 ml), saturated sodiumbicarbonate, and brine, dried, and concentrated to give the product(14.5 g, 100%). MS m/e 306 (M+H)⁺

To an ice-cold solution of triphenylphosphine (13.95 g, 53.19 mmol) inTHF (400 ml) and CH₃CN (50 ml) was added DIAD (10.76 g, 53.21 mmol). Themixture was stirred for 15 min and a solution of the product of Step 4(8.20 g, 26.2 mmol) in THF (100 ml) was added over 15 min. After theaddition was complete, the ice-water bath was removed and the mixturewas stirred at RT for 2 d. The mixture was concentrated and purified bycolumn chromatography (gradient EtOAc/Hexanes 0-5%) to give the product(3.75 g, 50%). MS m/e 288 (M+H)⁺

To a suspension of 60% NaH (6.40 g, 0.160 mol) in anhydrous DMA (400 ml)was added allyl alcohol (8.90 g, 0.154 mol) slowly. The mixture wasstirred at RT for 1 h. 3,5-Difluorobromobenzene (30.0 g, 0.155 mol) wasadded and the mixture was stirred at RT for 24 h. The reaction wasquenched with water (1.5 l) and extracted with ether (4×300 ml). Thecombined organic layer was washed with brine (500 ml), dried (MgSO₄),concentrated, and purified by column chromatography (Hexanes) to givethe product (14.3 g, 40%). ¹H-NMR (CDCl₃): δ=6.81 (m, 2H), 6.53 (m, 1H),5.96 (m, 1H), 5.34 (m, 2H), 4.46 (m, 2H).

To a flame-dried flask was added magnesium turnings (292 mg, 12.0 mmol)followed by one third of a solution of the product of Step 6 (2.31 g,10.0 mmol) in THF (16 ml). The reaction was initiated with dibromoethane(50 μl) then the remaining solution of the product of Step 6 was addedslowly. The mixture was stirred at RT for 30 min and added to asuspension of CuBr-Me₂S (310 mg, 1.51 mmol) in THF (30 ml) at −40° C.The mixture was stirred at 4° C. for 30 min and a solution of theproduct of Step 5 (1.20 g, 4.17 mmol) in anhydrous ether (15 ml) wasadded. The resulting mixture was stirred at 4° C. for 1 h then at RT for3 d. The reaction was quenched with saturated NH₄Cl (100 ml) andextracted with EtOAc (2×150 ml). The combined organic layer was washedwith saturated sodium bicarbonate and brine, dried (MgSO₄),concentrated, and purified by column chromatography (gradientEtOAc/Hexanes 0-5%) to give the product (1.00 g, 55%). MS m/e 440 (M+H)⁺

A solution of the product of Step 7 (500 mg, 1.14 mmol) in CH₂Cl₂ (12ml) and 4N HCl/dioxane (6 ml) was stirred at RT for 20 h. The mixturewas concentrated and the residue was partitioned between CH₂Cl₂ (50 ml)and 5N NH₄OH (20 ml). The organic layer was dried (K₂CO₃) andconcentrated to give the product (345 mg, 100%). MS m/e 226 (M+H)⁺

A mixture of the product of Step 8 (445 mg, 1.98 mmol), the product ofStep 2 (539 mg, 2.18 mmol), HOBt (442 mg, 3.27 mmol), and EDCI (627 mg,3.27 mmol) in DMF (20 ml) was stirred at RT for 3 d. The mixture wasconcentrated and the residue was partitioned between CH₂Cl₂ (200 ml) and1N NaOH. The organic layer was washed with 5% citric acid and brine,dried (MgSO₄), and concentrated. A solution of the residue in MeOH (20ml) and 1N NaOH (10 ml) was stirred at RT for 4 h. The mixture wasconcentrated and the residue was partitioned between EtOAc (150 ml) andsaturated NaHCO₃. The organic layer was washed with brine, dried(MgSO₄), concentrated, and purified by column chromatography (gradientMeOH/CH₂Cl₂ 0-2.5%) to give the product (405 mg, 45%). ¹H-NMR (CDCl₃):δ=7.63 (m, 2H), 7.37 (m, 1H), 7.28 (m, 1H), 7.07 (m, 1H), 6.56 (m, 2H),6.43 (m, 1H), 5.95 (m, 1H), 5.5-5.9 (m, 1H), 5.33 (m, 1H), 5.0-5.25 (m,3H), 4.42 (m, 2H), 4.23 (m, 1H), 4.07 (m, 1H), 3.5-3.8 (m, 3H), 3.36 (m,2H), 3.06 (m, 1H), 2.86 (m, 2H), 1.3-1.7 (m, 2H), 0.6-1.0 (m, 3H).

A mixture of the product of Step 9 (400 mg, 0.880 mmol) and 2^(nd)generation Grubb's catalyst (37 mg, 0.044 mmol) in CH₂Cl₂ (200 ml) washeated at 50° C. for 2.5 h then at RT for 16 h. The mixture wasconcentrated and purified by column chromatography (gradient MeOH/CH₂Cl₂0-2.5%) to give the product (340 mg, 91%). MS m/e 427 (M+H)⁺

A mixture of the product of Step 10 (340 mg, 0.797 mmol), ammoniumformate (50 mg, 0.79 mmol), and 10% Pd/C (50 mg) was stirred under H₂ (1atm) for 18 h. The mixture was filtered, concentrated, and used withoutfurther purification. A mixture of this material (129 mg, 0.301 mmol)and Dess-Martin periodinane (510 mg, 1.20 mmol) in CH₂Cl₂ (25 ml) wasstirred at RT for 2 h. The reaction was quenched with 1N Na₂S₂O₃ andpartitioned between saturated NaHCO₃ and CH₂Cl₂ (100 ml). The organiclayer was washed with brine, dried (MgSO₄), and concentrated to give thecrude aldehyde.

To a solution of the product of Preparative Example 1, Step 8 (261 mg,0.900 mmol) in anhydrous THF (5 ml) in a dry ice-acetone bath was added2M LDA (0.45 ml) and the mixture was stirred for 1 h. A solution of theabove aldehyde in THF (5 ml) was added and the mixture was stirred inthe dry ice-acetone bath for 1 h. The reaction was quenched withsaturated NH₄Cl (5 ml) and diluted with CH₂Cl₂ (50 ml). The organiclayer was washed with water and brine, dried (MgSO₄), concentrated, andpurified by PTLC (5% MeOH/CH₂Cl₂) to give the product (120 mg, 56%). MSm/e 717 (M+H)⁺

A mixture of the product of Step 11 (120 mg, 0.167 mmol) and TFA (2 ml)in CH₂Cl₂ (8 ml) was stirred at RT for 1.5 h. The mixture wasconcentrated and purified by PTLC (5% MeOH/CH₂Cl₂) to give:

fraction A (22 mg, 21%). ¹H-NMR (CDCl₃): δ=7.05-7.8 (m, 9H), 6.7-7.0 (m,1H), 6.50 (m, 2H), 6.36 (m, 1H), 4.4-5.0 (m, 3H), 3.9-4.3 (m, 2H),3.4-3.9 (m, 3H), 2.7-3.4 (m, 9H), 1.2-2.1 (m, 8H), 0.94 (m, 2H), 0.59(m, 1H). MS m/e 617 (M+H)⁺

fraction B (17 mg, 16%). ¹H-NMR (CDCl₃): δ=7.45-7.65 (m, 2H), 7.1-7.3(m, 8H), 6.95 (m, 1H), 6.56 (m, 1H), 6.37 (m, 1H), 4.68 (m, 2H), 3.9-4.4(m, 3H), 2.7-3.9 (m, 13H), 1.2-2.2 (m, 7H), 1.04 (m, 2H), 0.58 (m, 1H).MS m/e 617 (M+H)⁺

fraction C (12 mg, 12%). ¹H-NMR (CDCl₃): δ=7.2-7.8 (m, 8H), 6.75-7.0 (m,1H), 6.5-6.7 (m, 2H), 62-6.4 (m, 2H), 4.4-4.8 (m, 3H), 3.6-4.2 (m, 3H),2.7-3.4 (m, 10H), 1.2-2.2 (m, 8H), 0.95 (m, 2H), 0.61 (m, 1H). MS m/e617 (M+H)⁺

PREPARATIVE EXAMPLE 3

A mixture of itaconic acid (13.0 g, 100.0 mmol) and allyl amine (5.71 g,100 mmol) in anhydrous toluene (100 ml) was heated in a sealed tube at125° C. for 16 h. After the mixture was cooled down to RT, 1N aqueousNaOH (400 ml) was added and the aqueous layer was extracted with ether(2×200 ml). The aqueous layer was acidified with conc. HCl to pH 1 andextracted with ether (10×300 ml). The combined organic portion wasconcentrated and the residue was dissolved with CH₂Cl₂ (200 ml) andwashed with brine. The organic layer was dried with MgSO₄, concentrated,and lyophilized to give a light yellow solid (9.60 g, 57%). MS m/e 170(M+H)⁺

To a solution of the product of Step 1 (8.60 g, 50.9 mmol) andtriethylamine (15.4 g, 153 mmol) in anhydrous THF (200 ml) at −45° C.was added pivaloyl chloride (6.45 g, 53.5 mmol). The mixture was stirredat −45° C. for 1 h and then added into a suspension of lithium chloride(4.75 g, 112 mmol) and (S)-4-benzyl-2-oxazolidinone (9.02 g, 50.9 mmol)in THF (100 ml). The resulting mixture was stirred at RT for 16 h andfiltered. The filtrate was concentrated, dissolved in EtOAc (700 ml),and washed with 1N HCl (200 ml), saturated sodium bicarbonate (200 ml),and brine. The organic layer was dried (MgSO₄), concentrated, andpurified by column chromatography (gradient 0-75% EtOAc/Hexanes) to givethe product (7.20 g, 43%). MS m/e 329 (M+H)⁺

To a solution of the product of Step 2 (2.63 g, 8.01 mmol) in THF (30ml) and water (8 ml) in an ice-water bath were added 30% hydrogenperoxide (4 ml) and lithium hydroxide (0.672 g, 16.0 mmol). The mixturewas stirred at 0° C. for 7 h. 10% Aqueous sodium bisulfite (40 ml) wasadded and the mixture was stirred at RT for 16 h. The mixture wasconcentrated and the residue was partitioned between 1N NaOH (8 ml) andCH₂Cl₂ (2×100 ml). The aqueous layer was acidified to pH 2 at 0° C. andextracted with ether (5×100 ml). The combined organic portion was dried(MgSO₄) and concentrated to give the product (1.00 g, 74%). MS m/e 170(M+H)⁺

A solution of the Preparative Example 2, Step 8 (3.10 g, 13.0 mmol) andpotassium carbonate (5.39 g, 39.0 mmol) in EtOH (30 ml) and water (90ml) was heated to 70° C. Benzyl bromide (3.42 ml, 28.6 mmol) was addedand the mixture was stirred at 70° C. for 2.5 h. EtOH was removed andthe residue was extracted with ether (2×200 ml). The organic layer waswashed with brine, dried (K₂CO₃), concentrated, and purified by columnchromatography (gradient 0-10% EtOAc/Hexanes) to give the product (4.40g, 83%). MS m/e 406 (M+H)⁺

To a solution of oxalyl chloride (762 mg, 6.00 mmol) in CH₂Cl₂ (10 ml)in a dry ice-acetone bath was added DMSO (938 mg, 12.0 mmol). After 5min, a solution of the product of Step 4 (2.03 g, 5.01 mmol) in CH₂Cl₂(20 ml) was added and the mixture was stirred for 1 h. Triethylamine(2.42 g, 23.9 mmol) was added and after 2 min the cooling bath wasremoved. The mixture was stirred for 30 min and diluted with water (50ml). CH₂Cl₂ (100 ml) was added and the aqueous layer was extracted withCH₂Cl₂ (2×100 ml). The combined organic layer was washed with brine,dried (MgSO₄), and concentrated to give the aldehyde, which was notfurther purified.

To a solution of diisopropylamine (667 mg, 6.59 mmol) in THF (5 ml) in adry ice-acetone bath was added 1.6 M butyllithium in hexanes (4.13 ml,6.61 mmol). After 5 min the mixture was put in an ice-water bath andstirred for 20 min. The solution was cooled in the dry ice-acetone bathagain and a solution of the product of Preparative Example 1, Step 8(1.74 g, 5.99 mmol) in THF (20 ml) was added. The mixture was stirredfor 1 h. A solution of the above aldehyde in THF (30 ml) was added andthe mixture was allowed to warm up to RT slowly and stirred for 16 h.The reaction was quenched with saturated NH₄Cl (20 ml) and extractedwith ether (3×100 ml). The combined organic layer was washed with 5%citric acid, saturated NaHCO₃, and brine, dried (Na₂SO₄), concentrated,and purified by column chromatography (gradient EtOAc/Hexanes 0-40%) togive the product (1.20 g, 35%). MS m/e 694 (M+H)⁺

A solution of the product of Step 5 (1.00 g, 1.44 mmol) in MeOH (15 ml)was degassed with N₂. Anhydrous potassium carbonate (594 mg, 4.30 mmol)and Pd(PPh₃)₄ (324 mg, 0.280 mmol) were added and the mixture wasstirred at RT for 5 h. The mixture was filtered and concentrated. Theresidue was dissolved in CH₂Cl₂ (150 ml) and washed with 5% citric acid,saturated NaHCO₃, and brine. The organic layer was dried (MgSO₄) andconcentrated to give the product (980 mg, 100%). MS m/e 654 (M+H)⁺

A mixture of the product of Step 6 (980 mg, 1.44 mmol), 20% Pd(OH)₂/C(980 mg), and AcOH (1 ml) in EtOH (50 ml) was stirred under H₂ (1 atm)for 12 h. The mixture was filtered and concentrated to give the product(682 mg, 100%). MS m/e 474 (M+H)⁺

A mixture of the product of Step 7 (66 mg, 0.14 mmol), the product ofStep 3 (28 mg, 0.17 mmol), HOBt (38 mg, 0.28 mmol), EDCI (53 mg, 0.28mmol), and triethylamine (40 μl, 0.28 mmol) in CH₂Cl₂ (5 ml) was stirredat RT for 22 h. 1N NaOH (10 ml) was added and the mixture was stirredfor 30 min. The mixture was diluted with CH₂Cl₂ (50 ml), washed with 5%citric acid, water and brine, dried (MgSO₄), concentrated, and purifiedby PTLC (5% MeOH/CH₂Cl₂) to give the product (66 mg, 76%). MS m/e 625(M+H)⁺

A mixture of the product of Step 8 (66 mg, 0.11 mmol), potassiumcarbonate (146 mg, 1.06 mmol), and allyl bromide (14 mg, 0.12 mmol) inacetone (4 ml) was stirred at RT for 18 h. The mixture was partitionedbetween 5% citric acid and CH₂Cl₂ (50 ml). The organic layer was washedwith saturated NaHCO₃ and brine, dried (MgSO₄), concentrated, andpurified by PTLC (5% MeOH/CH₂Cl₂) to give the product (40 mg, 57%). MSm/e 665 (M+H)⁺

To a solution of the product of Step 9 (40 mg, 0.060 mmol) in CH₂Cl₂ (20ml) was added the 2^(nd) generation Grubb's catalyst (5 mg, 0.006 mmol).The mixture was degassed for 5 min with N₂ and then heated to 50° C. for2 h. The mixture was concentrated and purified by PTLC (5% MeOH/CH₂Cl₂)to give the product (30 mg, 79%). MS m/e 637 (M+H)⁺

A mixture of the product of Step 10 (30 mg, 0.047 mmol) and 10% Pd/C (30mg) in EtOH (5 ml) was stirred under H₂ (1 atm) for 75 min. The mixturewas filtered and concentrated to give the product (30 mg, 100%). MS m/e639 (M+H)⁺

A solution of the product of Step 11 (30 mg, 0.047 mmol) and TFA (1 ml)in CH₂Cl₂ (4 ml) was stirred at RT for 1.5 h. The mixture wasconcentrated and purified by PTLC (5% 2M NH₃/MeOH-95% CH₂Cl₂) to givethe product (20 mg, 79%). ¹H-NMR (CDCl₃): δ=7.25-7.35 (m, 5H), 6.53 (m,1H), 6.42 (m, 2H), 4.67 (d, 1H, J=14.4 Hz), 4.44 (d, 1H, J=14.4 Hz),4.36 (m, 1H), 3.8-4.2 (m, 4H), 3.60 (m, 1H), 2.8-3.4 (m, 9H), 2.69 (m,1H), 2.54 (m, 2H), 1.84 (m, 2H), 1.66 (m, 1H), 1.47 (m, 2H). LCMSt_(R)=2.88 min m/e 539 (M+H)⁺

PREPARATIVE EXAMPLE 4

To a solution of the product of step 7 in Preparative Example 3 (236 mg,0.5 mmol, crude product from previous step) in anhydrous THF (20 mL) wasadded 2.0 mL of 2 M borane-dimethylsufide in THF at RT. The mixture washeated at 60-70° C. overnight. After addition of MeOH (30 mL), themixture was heated at 70° C. for additional 2 h. Cooled to RT andconcentrated to dryness in a rotavapor. Additional MeOH (30 mL) wasadded and concentrated to dryness. The product was used directly in thenext step without further purification. MS m/e 460 (M+H)⁺

A solution of the product of Step 3 in Preparative Example 3 (102 mg,0.60 mmol), HOBt (162 mg, 1.2 mmol), and EDCI (230 mg, 1.2 mmol) inCH₂Cl₂ (6 ml) was stirred at RT for 1.5 h. Then a solution of theproduct of Step 1 (crude, ˜0.5 mmol) in CH₂Cl₂ (20 mL) was added,followed by triethylamine (240 mg, 2.4 mmol). The mixture was stirred atRT for 22 h. 1N NaOH (20 ml) was added and the mixture was stirred for45 min. The mixture was diluted with CH₂Cl₂ (150 ml), washed with H₂O,5% citric acid, and brine, dried (MgSO₄), concentrated, and purified byISCO (elution with CH₂Cl₂ for 10 min., 0-2.5% MeOH/CH₂Cl₂ for 35 min.)to give the product (90 mg, 29% for 3 steps). ¹H-NMR (CDCl₃):δ=7.22-7.32 (m, 5H), 6.58 (d, 1H, J=8.8 Hz), 6.39 (m, 2H), 6.25 (m, 1H),3.79-3.93 (m, 5H), 3.27-3.58 (m, 7H), 3.06 (d, 1H, J=10 Hz), 2.96 (m,1H), 2.77 (d, 1H, 10.8 Hz), 2.50-2.56 (m, 2H), 2.05-2.32 (m, 4H), 1.38(s, 9H). MS m/e 611 (M+H)⁺.

A mixture of the product of Step 3 (40 mg, 0.066 mmol), potassiumcarbonate (91 mg, 0.66 mmol), and allyl bromide (18 mg, 0.15 mmol) inacetone (5 ml) was at 60-70° C. for 18 h. Cooled to RT, filtered andconcentrated. The residue was dissolved in CH₂Cl₂ (50 ml). The organiclayer was washed with brine and dried (MgSO₄), concentrated, andpurified by PTLC (5% MeOH/CH₂Cl₂) to give the product (27 mg, 63%).¹H-NMR (CDCl₃): δ=7.22-7.30 (m, 5H), 6.41 (m, 3H), 5.96 (m, 1H), 5.84(d, 1H, J=8.9 Hz), 5.62 (m, 1H), 5.37 (dd, 1H, J=1.6 Hz), 5.33 (dd, 1H,J=1.6 Hz), 5.23 (dd, 1H, J=1.2 Hz, J=10 Hz), 5.09-5.14 (m, 2H), 4.43(dt, 2H, J=6.4 Hz, J=1.5 Hz), 3.69-3.95 (m, 5H), 3.26-3.58 (m, 7H), 3.08(d, 1H, J=10 Hz), 2.92 (m, 1H), 2.77 (d, 1H, J=11 Hz), 2.63 (m, 1H),2.42 (m, 2H), 2.22 (d, 1H, J=9 Hz), 2.06 (m, 1H), 1.37 (s, 9H). MS m/e651 (M+H)⁺

To a solution of the product of Step 3 (60 mg, 0.092 mmol) in CH₂Cl₂ (40ml) was added the 2^(nd) generation Grubb's catalyst (7.8 mg, 0.0092mmol). The mixture was degassed for 5 min with N₂ and then heated to40-50° C. for 1 h. The mixture was concentrated and purified by PTLC (5%MeOH/CH₂Cl₂) to give the product (52 mg, 91%). ¹H-NMR (CDCl₃):δ=7.22-7.32 (m, 5H), 6.51 (d, 1H, J=8.8 Hz), 6.40 (d, 1H, J=10 Hz), 6.32(s, 1H), 6.26 (d, 1H, J=8.8 Hz), 5.65 (m, 2H), 4.60 (s, 2H), 4.56 (d,1H, J=3.2 Hz), 4.00 (s, 1H), 3.85 (d, 2H, J=10 Hz), 3.17-3.51 (m, 6H),2.62-2.97 (m, 6H), 2.47 (t, 1H, J=12 Hz), 2.05-2.15 (m, 2H), 1.78-1.88(m, 2H), 1.41 (s, 9H). MS m/e 623 (M+H)⁺.

A mixture of the product of Step 4 (52 mg, 0.084 mmol) and 20% Pd(OH)₂/C(52 mg) in EtOH (5 ml) and acetic acid (0.4 mL) was stirred under H₂ (1atm) for 90 min. The mixture was filtered and concentrated. The residuewas dissolved in CH₂Cl₂ (50 mL) and washed with saturated NaHCO₃ andbrine, then dried (K₂CO₃) and concentrated to give the product (33 mg,74%). MS m/e 535 (M+H)⁺.

To an ice-cooled solution of the product of step 5 (33 mg, 0.062 mmol)in anhydrous CH₂Cl₂ (6 mL) was added triethylamine (20 mg), followed bya solution of m-toluenesulfonyl chloride (13 mg, 0.068 mmol) in CH₂Cl₂.The mixture was stirred at 0° C. for 1 h. Then diluted with CH₂Cl₂ (50mL), washed with 5% of citric acid, saturated NaHCO₃ and brine, anddried (MgSO₄). The solution was concentrated and purified by PTLC (5%MeOH/CH₂Cl₂) to give the product (27 mg, 63%). MS m/e 689 (M+H)⁺.

A solution of the product of Step 6 (27 mg, 0.039 mmol) and TFA (1 ml)in CH₂Cl₂ (4 ml) was stirred at RT for 1 h. The mixture was concentratedand purified by PTLC (5% 2M NH₃/MeOH-95% CH₂Cl₂) to give the product (23mg, 94%). ¹H-NMR (CDCl₃): δ=7.49 (m, 2H), 7.38 (m, 2H), 6.53 (d, 1H, J=8Hz), 6.45 (s, 1H), 6.39 (m, 1H), 4.01-4.17 (m, 3H), 3.82-3.85 (m, 2H),3.64 (m, 1H), 3.23-3.43 (m, 3H), 2.85-3.10 (m, 7H), 2.66-2.70 (m, 2H),2.50-2.57 (m, 2H), 2.40 (s, 3H), 1.88 (s, broad, 1H), 1.65 (m, 1H),1.39-1.47 (m, 2H). LCMS t_(R)=2.99 min m/e 589 (M+H)⁺

BACE-1 Cloning, Protein Expression and Purification

A predicted soluble form of human BACE1 (sBACE1, corresponding to aminoacids 1-454) was generated from the full length BACE1 cDNA (full lengthhuman BACE1 cDNA in pcDNA4/mycHisA construct; University of Toronto) byPCR using the advantage-GC cDNA PCR kit (Clontech, Palo Alto, Calif.). AHindIII/PmeI fragment from pcDNA4-sBACE1myc/His was blunt ended usingKlenow and subcloned into the Stu I site of pFASTBACI(A) (Invitrogen). AsBACE1mycHis recombinant bacmid was generated by transposition inDH10Bac cells (GIBCO/BRL). Subsequently, the sBACE1mycHis bacmidconstruct was transfected into sf9 cells using CellFectin (Invitrogen,San Diego, Calif.) in order to generate recombinant baculovirus. Sf9cells were grown in SF 900-II medium (Invitrogen) supplemented with 3%heat inactivated FBS and 0.5× penicillin/streptomycin solution(Invitrogen). Five milliliters of high titer plaque purifiedsBACEmyc/His virus was used to infect 1 L of logarithmically growing sf9cells for 72 hours. Intact cells were pelleted by centrifugation at3000×g for 15 minutes. The supernatant, containing secreted sBACE1, wascollected and diluted 50% v/v with 100 mM HEPES, pH 8.0. The dilutedmedium was loaded onto a Q-sepharose column. The Q-sepharose column waswashed with Buffer A (20 mM HEPES, pH 8.0, 50 mM NaCl).

Proteins, were eluted from the Q-sepharose column with Buffer B (20 mMHEPES, pH 8.0, 500 mM NaCl). The protein peaks from the Q-sepharosecolumn were pooled and loaded onto a Ni-NTA agarose column. The Ni-NTAcolumn was then washed with Buffer C (20 mM HEPES, pH 8.0, 500 mM NaCl).Bound proteins were then eluted with Buffer D (Buffer C+250 mMimidazole). Peak protein fractions as determined by the Bradford Assay(Biorad, Calif.) were concentrated using a Centricon 30 concentrator(Millipore). sBACE1 purity was estimated to be ˜90% as assessed bySDS-PAGE and Commassie Blue staining. N-terminal sequencing indicatedthat greater than 90% of the purified sBACE1 contained the prodomain;hence this protein is referred to as sproBACE1.

Peptide Hydrolysis Assay

The inhibitor, 25 nM EuK-biotin labeled APPsw substrate(EuK-KTEEISEVNLDAEFRHDKC-biotin; CIS-Bio International, France), 5 μMunlabeled APPsw peptide (KTEEISEVNLDAEFRHDK; American Peptide Company,Sunnyvale, Calif.), 7 nM sproBACE1, 20 mM PIPES pH 5.0, 0.1% Brij-35(protein grade, Calbiochem, San Diego, Calif.), and 10% glycerol werepreincubated for 30 min at 30° C. Reactions were initiated by additionof substrate in a 5 μl aliquot resulting in a total volume of 25 μl.After 3 hr at 30° C. reactions were terminated by addition of an equalvolume of 2× stop buffer containing 50 mM Tris-HCl pH 8.0, 0.5 M KF,0.001% Brij-35, 20 μg/ml SA-XL665 (cross-linked allophycocyanin proteincoupled to streptavidin; CIS-Bio International, France) (0.5 μg/well).Plates were shaken briefly and spun at 1200×g for 10 seconds to pelletall liquid to the bottom of the plate before the incubation. HTRFmeasurements were made on a Packard Discovery® HTRF plate reader using337 nm laser light to excite the sample followed by a 50 μs delay andsimultaneous measurements of both 620 nm and 665 nm emissions for 400μs.

IC₅₀ determinations for inhibitors, (I), were determined by measuringthe percent change of the relative fluorescence at 665 nm divided by therelative fluorescence at 620 nm, (665/620 ratio), in the presence ofvarying concentrations of I and a fixed concentration of enzyme andsubstrate. Nonlinear regression analysis of this data was performedusing GraphPad Prism 3.0 software selecting four parameter logisticequation, that allows for a variable slope.Y=Bottom+(Top-Bottom)/(1+10^((LogEC50−X)*Hill Slope)); X is thelogarithm of concentration of I, Y is the percent change in ratio and Ystarts at bottom and goes to top with a sigmoid shape.

Compounds of the present invention have an IC₅₀ range from about 100 toabout 10,000 nM, preferably about 100 to about 1000 nM, more preferablyabout 100 to about 500 nM. Compounds of the preferred stereochemistryhave IC₅₀ values in a range of about 2 to about 500 nM, preferably about2 to about 100 nM. The compound of the following formula

has an IC₅₀ of 4 nM.The following table demonstrates the IC₅₀ classifications for thefollowing compounds.

Compounds with an IC₅₀ of 2 to 1000 nM are A class compounds.

Compounds with an IC₅₀ of 1000 to 10000 nM are B class compounds.

Compounds with an IC₅₀ over 10000 nM are C class compounds.

COMPOUND Activity

B

A or B

A

While the present invention has been described in conjunction with thespecific embodiments set forth above, many alternatives, modificationsand variations thereof will be apparent to those of ordinary skill inthe art. All such alternatives, modifications and variations areintended to fall within the spirit and scope of the present invention.

1. A compound having the structural formula

or a pharmaceutically acceptable salt thereof, wherein R¹ is

R² is —N(R⁵)C(O)R⁴—; R³ is arylene, heteroarylene, heterocyclylene orcycloalkylene; R⁴ is arylene, heteroarylene, heterocyclylene orcycloalkylene; R⁵ is hydrogen, alkyl, aryl, heteroaryl or cycloalkyl; R⁶and R⁷ (when present) are independently selected from hydrogen, —OH,alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, aryl, heteroaryl,aralkyl, heteroaralkyl, aralkoxy, heteroaralkoxy and alkoxy, with theproviso that when R⁶ and R⁷ are —OH, aralkoxy, heteroaralkoxy andalkoxy, R⁶ and R⁷ are not attached to a ring carbon adjacent to a ringnitrogen; R⁸ is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl,heteroaryl, aralkyl, heteroaralkyl, heterocyclyl, heterocyclylalkyl,—C(O)R⁹, —C(O)OR¹², —S(O)R⁹, —S(O₂)R⁹ or —CN; with the proviso that whenY is O, R⁸ cannot be —C(O)R⁹, —C(O)OR¹², —S(O)R⁹, —S(O₂)R⁹ or —CN; R⁹ ishydrogen, alkyl, cycloalkyl, aryl, heteroaryl, cycloalkylalkyl, aralkyl,heteroaralkyl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl or—N(R¹⁰)(R¹¹); R¹⁰ and R¹¹ are independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl,heteroaryl, heterocyclyl, aralkyl, heteroaralkyl, heterocyclylalkyl,alkenyl and alkynyl; or R¹⁰ and R¹¹ together with the nitrogen to whichthey are attached, form a 3-7 membered heterocyclyl ring; R¹² is alkyl,cycloalkyl, aryl, heteroaryl, cycloalkylalkyl, aralkyl, heteroaralkyl,heterocyclyl, heterocyclylalkyl, alkenyl or alkynyl; X is O, S, C(R⁵),or NH; Y is O, or (H,H); m is 1, 2, or 3; n is 0, 1, 2, or 3; and o is0, 1, 2, or 3; wherein each alkyl is optionally substituted with 1 to 3moieties selected from the group consisting of halo, aryl, cycloalkyl,cyano, hydroxy, alkoxy, alkylthio, amino, —NH(alkyl), —NH(cycloalkyl),—N(alkyl)₂, carboxy and —C(O)O-alkyl; and wherein each arylene,heteroarylene, heterocyclyl, heterocyclylalkyl, heterocyclylene,cycloalkylene, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl,heterocyclyl, aralkyl, heteroaralkyl, aralkoxy or heteroaralkoxy isoptionally substituted with 1 to 4 moieties selected from the groupconsisting of —CF₃, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl,alkylaryl, heteroaralkyl, heteroarylalkenyl, heteroarylalkynyl,alkylheteroaryl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, acyl,aroyl, halo, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl,aralkoxycarbonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl,alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio,cycloalkyl, heterocyclyl, —C(═N—CN)—NH₂, —C(═NH)—NH₂, —C(═NH)—NH(alkyl),Y₁Y₂N—, Y₁Y₂N-alkyl-, Y₁Y₂NC(O)—, Y₁Y₂NSO₂— and —SO₂NY₁Y₂, wherein Y₁and Y₂ can be the same or different and are independently selected fromthe group consisting of hydrogen, alkyl, aryl, cycloalkyl, and aralkyl,with the proviso that cycloalkylene and heterocyclylene can besubstituted with ═O.
 2. The compound of claim 1 wherein R¹ is


3. The compound of claim 1 wherein R³ is arylene.
 4. The compound ofclaim 3 wherein R³ is phenylene or halo-substituted phenylene.
 5. Thecompound of claim 3 wherein R³ is


6. The compound of claim 3 wherein R³ is


7. The compound of claim 1 wherein R⁴ is arylene.
 8. The compound ofclaim 1 wherein R⁴ is phenylene.
 9. The compound of claim 1 wherein R⁴is


10. The compound of claim 1 wherein R⁵ is alkyl.
 11. The compound ofclaim 10 wherein R⁵ is propyl.
 12. The compound of claim 1 wherein m is2 and n is
 1. 13. The compound of claim 1 wherein R⁷ is hydrogen. 14.The compound of claim 1 wherein X is O.
 15. The compound of claim 1wherein Y is O.
 16. The compound of claim 1 wherein R⁸ is aralkyl or—S(O₂)R⁹.
 17. The compound of claim 1 wherein R⁸ is


18. The compound of claim 1 wherein R¹ is

R² is —N(R⁵)C(O)R⁴—; R³ is arylene R⁴ is arylene or heterocyclylene; R⁵is alkyl; R⁷ is hydrogen; R⁸ is aralkyl or —S(O₂)R⁹; m is 2; n is 1; Xis O; and Y is O.
 19. The compound of claim 18 wherein R³ is phenyleneor halo-substituted phenylene.
 20. The compound of claim 19 wherein R³is


21. The compound of claim 20 wherein R³ is


22. The compound of claim 18 wherein R⁴ is arylene.
 23. The compound ofclaim 18 wherein R⁴ is


24. The compound of claim 18 wherein R² is heterocyclylene orheterocyclylene substituted with ═O.
 25. The compound of claim 18wherein R⁵ is propyl.
 26. The compound of claim 18 wherein R⁸ is


27. A compound of claim 1 having the stereochemical structure


28. A compound of claim 1 selected from the group consisting of


29. A pharmaceutical composition comprising an effective amount of acompound of claim 1 and a pharmaceutically effective carrier.
 30. Apharmaceutical composition comprising an effective amount of a compoundof claim 1, and an effective amount of a cholinesterase inhibitor in apharmaceutically effective carrier.